Abstract: P2X7 receptor/channels in the retinal microvasculature not only regulate vasomotor activity, but can also trigger cells in the capillaries to die. While it is known that this purinergic vasotoxicity is dependent on the transmembrane pores that form during P2X7 activation, events linking pore formation with cell death remain uncertain. To better understand this pathophysiological process, we used YO-PRO-1 uptake, dichlorofluorescein fluorescence, perforated-patch recordings, fura-2 imaging and trypan blue dye exclusion to assess the effects of the P2X7 agonist, benzoylbenzoyl-ATP (BzATP), on pore formation, oxidant production, ion channel activation, [Ca2+ ]i and cell viability. Experiments demonstrated that exposure of retinal microvessels to BzATP increases capillary cell oxidants via a mechanism dependent on pore formation and the enzyme, NADPH oxidase. Indicative that oxidation plays a key role in purinergic vasotoxicity, an inhibitor of this enzyme completely prevented BzATP-induced death. We further discovered that vasotoxicity was boosted 4-fold by a pathway involving the oxidation-driven activation of hyperpolarizing KATP channels and the resulting increase in calcium influx. Our findings revealed that the previously unappreciated pore/oxidant/KATP channel/Ca2+ pathway accounts for 75% of the capillary cell death triggered by sustained activation of P2X7 receptor/channels. Elucidation of this pathway is of potential therapeutic importance since purinergic vasotoxicity may play a role in sight-threatening disorders such as diabetic retinopathy. Keywords: KATP channels; oxidants; P2X7 ; pores; retina

1. Introduction Although functional P2X7 receptor/channels were discovered in retinal capillaries 15 years ago, much remains to be learned about their impact on retinovascular physiology and pathobiology. While it is clear that activation of P2X7 receptor/channels causes the abluminal pericytes of retinal capillaries to contract [1], the role of P2X7 -induced vasoconstriction in regulating retinal blood flow is uncertain. One possibility is that these purinergic receptor/channels transduce the putative glial-to-vascular signal, ATP [2], into an alteration in local perfusion. They may also play a role in mediating vascular responses to nicotinamide adenosine dinucleotide (NAD+ ), which is candidate signaling molecule [3] whose ribosylation activates P2X7 receptor/channels in retinal capillaries [4]. Not only do P2X7 receptor/channels induce vasomotor activity, but we also discovered that their sustained activation can trigger the death of capillary cells [4–6]. Noteworthy is that this toxic effect of vasoactive purinergic input is conceptually similar to the extensively studied phenomenon of the neurotransmitter glutamate being neurotoxic. Previously, we reported that an essential mechanistic Vision 2018, 2, 25; doi:10.3390/vision2030025

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step in purinergic vasotoxicity is the formation of large transmembrane pores [4–6], whose opening during P2X7 activation has been intensively analyzed in many types of cells [7,8]. In non-vascular tissue, such as cells of the immune system, these pores appear to cause death by severely disrupting ionic gradients and/or by providing pathways for the egress of vital intracellular molecules [9–12]. However, since pore formation is relatively limited in retinal capillary cells [6], it remains uncertain how the opening of pores triggers death. Thus, the quest of this study was to identify events that occur downstream from pore formation and that play key roles in mediating purinergic vasotoxicity. In this study using freshly isolated retinal microvascular complexes, we report that although the formation of P2X7 pores is required in order to trigger purinergic vasotoxicity in retinal capillaries, pore formation alone is not sufficient. Rather, a pore-dependent rise in intracellular oxidants is required. This study further revealed that the previously unappreciated pore/oxidant/KATP channel/Ca2+ pathway is the predominant mechanism mediating purinergic vasotoxicity in retinal capillaries. 2. Materials and Methods Experimental protocols for animal use were approved by the Institutional Animal Care and Use Committee of the University of Michigan and were consistent with the guidelines of the Association for Research in Vision and Ophthalmology for use of animals in ophthalmic and vision research. Male Long-Evans rats were obtained from Charles River (Cambridge, MA, USA). At all times, animals were kept on a 12-h alternating light/dark cycle and received food and water ad libitum. 2.1. Microvessel Isolation Using our previously described tissue print procedure [13], we isolated large complexes of microvessels from the retinas of adult rats. In brief, immediately after a rising concentration of carbon dioxide caused the death of a 6- to 14-week old rat, the retinas were removed and placed in solution A, which consisted of 140 mM NaCl, 3 mM KCl, 1.8 mM CaCl2 , 0.8 mM MgCl2 , 10 mM Na-Hepes, 15 mM mannitol, and 5 mM glucose at pH 7.4 with osmolarity adjusted to 310 mosmol L−1 , as measured by a vapor pressure osmometer (Wescor, Inc., Logan, UT, USA). After adherent vitreous was removed with fine forceps, each retina was cut into quadrants and incubated for 22 to 26 min at 30 ◦ C in 2.5 mL Earle’s balanced salt solution that was supplemented with 0.5 mM EDTA, 6 U papain (Worthington Biochemicals, Freehold, NJ, USA) and 2 mM cysteine; the pH was adjusted to approximately 7.4 by bubbling 5% carbon dioxide. After this incubation, the retinal pieces were transferred to a 60 mm Petri dish containing 5 mL of solution A, and one by one, each retinal quadrant was positioned with its vitreal surface up in a glass-bottomed chamber containing 1 mL of solution A. Subsequently, each retinal quadrant was gently sandwiched between the bottom of the chamber and a 15 mm diameter glass coverslip (Warner Instrument Corp., Hamden, CT, USA). After ~30 s, the coverslip was carefully removed; it contained adherent complexes of retinal microvessels. Microvascular complexes used in this study consisted of an arteriole encircled by “doughnut-shaped” myocytes, a tertiary vessel with ≥5 “dome-shaped” mural cell somas per 100 µm and a capillary network whose abluminal mural cells, the pericytes, appear as “bumps on a log” and have a density of ≤4 per 100 µm [13]. Photomicrographs of retinal microvessels isolated by this tissue print technique are available [13,14]. All measurements were obtained from the capillary portion. Experiments were completed within 7 h after microvessel isolation. 2.2. Imaging of Intracellular Oxidants To detect intracellular oxidants, microvessels were exposed for 30 min to solution A supplemented with the oxidant-sensitive dye, 6-carboxy-20 ,70 -dichlorodihydrofluorescein diacetate (carboxy-H2 DCFDA; 5 µM; Invitrogen, Eugene, OR, USA). The microvessel-containing coverslip was

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then positioned in a 200 µL recording chamber, which was perfused at ~1.5 mL min−1 via a gravity-fed system. Subsequent exposure to dye-free solution A for 10 min allowed carboxy-H2 DCFDA to be cleaved by intracellular esterases to carboxy-H2 DCF, which upon oxidation becomes fluorescent dichlorofluorescein (DCF). Microvessels were observed using a Nikon Eclipse TE300 microscope at X400 with a 40X water-immersion objective. The light source was a high intensity mercury lamp coupled to an Optoscan Monochromator (Cairn Research Ltd., Faversham, UK). Fluorescence was detected with excitation and emission wavelengths of 490 and 520 nm, respectively. Digital imaging of DCF fluorescence was performed using an optical sensor (Sensicam, Cooke Corp., Auburn Hills, MI, USA). Axon Imaging Workbench software (MDS Analytical Technologies) facilitated control of the imaging equipment and the collection of data. To minimize photo-oxidation, illumination was limited to 400-ms exposures at 30 s to 90 s intervals. Autofluroescence was not detected in microvessels not been exposed to carboxy-H2 DCFDA. In microvascular complexes exposed to carboxy-H2 DCFDA, both mural cells and endothelial cells became loaded with this dye. No attempt was made to selectively detect fluorescence in mural or endothelial cells; fluorescence from both cell types was detected. Regions of interest (ROIs) were selected on microvessels and in order to measure background fluorescence, on cell-free areas of the coverslip. Subtraction of background fluorescence from the intensity of fluorescence measured in microvascular ROIs yielded the net fluorescence. If FDCF varied by >5% during a 200-s control period in solution A, the microvascular was not further studied. For each group of ROIs, the control value was the average net fluorescence during the 200-s period prior to the onset of exposure to the P2X7 agonist, benzoylbenzoyl-ATP (BzATP, 100 µM). Net fluorescence measurements were plotted in Figure 2A as the percent of the control value. In Figures 2B and 4, net fluorescence increases were measured 600 s after the onset of BzATP exposure. In experiments using A740003 (200 nM), UTP (30 µM), apocynin (300 µM), n-acetyl-cysteine (NAC, 100 µM), glibenclamide (0.5 µM) or low calcium solution A (solution A without added CaCl2 ), pre-incubations prior to exposure to BzATP were at room temperature for 15 min, 30 min, 30 min, 60 min, 10 min and 30 min, respectively. 2.3. YO-PRO-1 Uptake To detect capillary cells containing transmembrane pores, microvascular complexes were exposed to propidium di-iodide dye, YO-PRO-1 (Molecular Probes, Eugene, OR, USA), which is a 629 Da molecule that becomes fluorescent after entering a cell and binding to nucleic acids. Initially, microvessels were exposed for 1 h at room temperature to solution A in the absence or in the presence of 30 µM UTP, 300 µM apocynin or 0.5 µM glibenclamide. Subsequently after addition of 100 µM BzATP to some experimental groups, microvessels were incubated for 4 h at 37 ◦ C and 100% humidity. Immediately after this incubation, bathing solutions were supplemented with 5 µM YO-PRO-1, and incubation was continued at 37 ◦ C for 30 min. After washout of the YO-PRO-containing solutions, microvessel-containing coverslips were positioned in a chamber located on the stage of a Nikon Eclipse TE300 or E800 microscope (Nikon, Tokyo, Japan) equipped for fluorescence and using X20 objectives. Differential interference contrast optics facilitated detection of cells lacking fluorescence. For each coverslip, more than 70 microvascular cells were examined, and the percentage of YO-PRO-positive cells was determined. It did not prove feasible to subclassify cells as endothelial or abluminal. The BzATP-induced increase in YO-PRO positive capillary cells was calculated by subtracting the percentage of positive cells observed in the appropriate control group from the BzATP-containing group. Notable is that in the absence of BzATP, neither 200 nM A740003, 30 µM UTP, 100 µM NAC, 0.5 µM glibenclamide nor the low calcium solution A significantly affected the percentage of YO-PRO positive cells, which in solution A alone was 9.9 ± 0.9% (n = 21) after 4 h at 37 ◦ C.

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2.4. Cell Viability Assay Microvascular cells that failed to exclude trypan blue were classified as dead. Microvessel-containing coverslips were exposed to 0.04% trypan blue assay in solution A for 15 min, and the percentage of trypan blue positive cells was determined by examining microvessel-containing coverslips at X100 magnification with an inverted microscope equipped with bright-field optics. Of note, because trypan blue-containing cells typically were swollen, identification of these cells as being endothelial or abluminal was uncertain, and thus, sub-classification of microvascular cells into these two types was not done in the cell death assays. As reported previously [6], the validity of this 961-Da vital stain was not compromised by the formation of P2X7 pores, which are permeable to molecules of ≤900 Da [15]. Prior to the onset of experimental conditions, cell viability was determined in microvascular complexes whose locations on each coverslip were carefully documented so that cell death could be re-assessed in the identical microvascular region. At least 150 microvascular cells per coverslip were counted. In experiments using A740003, UTP, apocynin, n-acetyl-cysteine or glibenclamide, microvessels were exposed to the additive in solution A for 1 h at room temperature. Subsequently, after the addition of BzATP, microvascular complexes were maintained for 6 h at 37 ◦ C and 100% humidity. Immediately after this incubation, the percentage of trypan blue capillary cells was again assayed for each of the microvascular regions assessed at time zero. The increase in cell death between time 0 and at 6 h was then calculated. Because the percentage of trypan blue capillary cells maintained for 6 h at 37 ◦ C increased from 4.4 ± 0.3% to 5.5 ± 0.2% (n = 23), the amount of cell death induced by BzATP was calculated by subtracting this 1.1% increase from the increase in trypan blue positive cells measured during BzATP exposure. 2.5. Electrophysiology The perforated-patch configuration of the patch clamp technique was used to detect ionic currents in microvascular complexes located on coverslips positioned in a recording chamber (V = 0.5 mL), which was perfused (~1.5 mL min−1 ) with solution A, which could be supplemented with 100 µM BzATP without or with 0.5 µM glibenclamide. Experiments were at 22 ◦ C to 23 ◦ C. Recording pipettes that were filled with a solution consisting of 50 mM KCl, 65 mM K2 SO4 , 6 mM MgCl2 , 10 mM K-Hepes, 60 µg mL−1 amphotericin B and 60 µg mL−1 nystatin at pH 7.4 with the osmolarity adjusted to 280 mosmol L−1 and that had resistances of 5 to 10 MΩ were mounted in the holder of a patch-clamp amplifier (Axopatch 200B, MDS Analytical Technologies, Union City, CA, USA) and were positioned onto the surface of capillary pericytes, which are easily identified by their “bump on the log” location [13,16]. Recordings with a seal resistance of ≥10 GΩ seal and an access resistance of 0.05 indicated failure to detect a significant difference. For greater than two groups, an analysis of variance was performed using commercially available software (Origin 2017) with the subsequent application of the Bonferroni correction. 3. Results The objective of this study was to elucidate how the activation of P2X7 receptor/channels can cause cells in retinal capillaries to die. To guide experimentation, we built upon previous studies to formulate a working model. As shown in Figure 1, the key instigating event in purinergic vasotoxicity is the formation of large transmembrane pores during sustained P2X7 activation [5]. Further, studies of non-retinal cells have demonstrated that the opening of P2X7 pores increases intracellular oxidants by a mechanism involving NADPH oxidase [22,23]. In addition, based on our previous analysis of the electrophysiological effects of the oxidant H2 O2 [24], we postulated that the P2X7 -induced increase in oxidants may activate redox-sensitive KATP channels whose voltage-increasing effect would enhance the electro-gradient for the influx of calcium whose elevated intracellular concentration is known to increase the lethality of oxidative stress [24,25]. Taken together, these considerations led us to posit that a pore/oxidant/KATP channel/Ca2+ pathway boosts the vulnerability of retinal capillaries to P2X7 vasotoxicity (Figure 1).

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Figure 1. Overview of the conceptual framework of this experimental study. Assays are shown in Figure 1. Overview of the conceptual framework of this experimental study. Assays are shown in green and inhibitors are in red with the dotted lines indicating sites of inhibition. UTP inhibits pore green and inhibitors are in red with the dotted lines indicating sites of inhibition. UTP inhibits pore formation by a mechanism involving P2Y4 receptors and phospholipase [5]. Low [Ca2+]o was solution formation by a mechanism involving P2Y receptors and phospholipase [5]. Low [Ca2+ ]o was solution 4 A without added calcium. NAC is the antioxidant, n‐acetyl‐cysteine. Although not the focus of this Figure 1. Overview of the conceptual framework of this experimental study. Assays are shown in A without added calcium. NAC is the antioxidant, n-acetyl-cysteine. Although not the focus of this 7 activation [1,26]. study, capillary vasoconstriction occurs with P2X green and inhibitors are in red with the dotted lines indicating sites of inhibition. UTP inhibits pore study, capillary vasoconstriction occurs with P2X7 activation [1,26]. formation by a mechanism involving P2Y4 receptors and phospholipase [5]. Low [Ca2+]o was solution To begin to assess the role of oxidants in purinergic vasotoxicity, the fluorescence of A without added calcium. NAC is the antioxidant, n‐acetyl‐cysteine. Although not the focus of this dichlorofluorescein (DCF) was used to detect a change in capillary cell oxidants during exposure to study, capillary vasoconstriction occurs with P2X To begin to assess the role of oxidants 7 activation [1,26]. in purinergic vasotoxicity, the fluorescence

of the P2X7 agonist, BzATP (100 μM). As illustrated in Figure 2A and summarized in Figure 2B, BzATP dichlorofluorescein (DCF) was used to detect a change in capillary cell oxidants during exposure to induced a significant increase in FDCF. Additional DCF experiments were performed in the presence To begin to assess the role of oxidants in purinergic vasotoxicity, the fluorescence of the P2Xof 200 nM A740003, which is a P2X agonist, BzATP (100 µM). As illustrated in Figure 2A and summarized in Figure 2B, BzATP 7 receptor antagonist [27] and 30 μM UTP, whose activation of 7 dichlorofluorescein (DCF) was used to detect a change in capillary cell oxidants during exposure to P2Y 4 inhibits P2X 7 pore formation in retinal microvessels [5]. We found that both A74003 and UTP induced a significant increase in FDCF . Additional DCF experiments were performed in the presence the P2X 7 agonist, BzATP (100 μM). As illustrated in Figure 2A and summarized in Figure 2B, BzATP markedly attenuated BzATP‐induced formation of pores and rise in capillary cell F (Figure 2B). of 200 nM A740003, which is a P2X7DCFreceptor antagonist [27] and 30 µM UTP, DCF whose activation of induced a significant increase in F . Additional DCF experiments were performed in the presence Indicative the BzATP‐induced oxidant production is not a rapidly reversible process, we observed of 200 nM A740003, which is a P2X 7 receptor antagonist [27] and 30 μM UTP, whose activation of P2Y4 inhibits P2X7 pore formation in retinal microvessels [5]. We found that both A74003 and UTP that FDCF increased by 9 ± 2% (n = 9; p = 0.0158) during the initial 5 min after the washout of BzATP. P2Yattenuated 4 inhibits P2X7 pore formation in retinal microvessels [5]. We found that both A74003 and UTP markedly BzATP-induced formation of7pores and rise in capillary cell FDCF (Figure 2B). Taken together, these results led us to conclude P2X activation triggers a sustained boost in the level markedly attenuated BzATP‐induced formation of pores and rise in capillary cell FDCF (Figure 2B). Indicative the BzATP-induced oxidant production is not a rapidly reversible process, we observed of oxidants in retinal capillary cells. Indicative the BzATP‐induced oxidant production is not a rapidly reversible process, we observed that FDCF increased by 9 ± 2% (n = 9; p = 0.0158) during the initial 5 min after the washout of BzATP. that FDCF increased by 9 ± 2% (n = 9; p = 0.0158) during the initial 5 min after the washout of BzATP. Taken together, these results led us to conclude P2X77 activation triggers a sustained boost in the level activation triggers a sustained boost in the level Taken together, these results led us to conclude P2X of oxidants in retinal capillary cells. of oxidants in retinal capillary cells.

Figure 2. Effect of the P2X7 agonist, BzATP, on intracellular oxidants, transmembrane pores and cell viability in capillaries located within microvascular complexes freshly isolated from the rat retina. (A) Fluorescence intensity of the oxidation‐sensitive dye, dichlorofluorescein (DCF), before and during exposure to 100 μM BzATP. Each data point is the mean of 9 regions of interest in retinal capillaries. Figure 2. Effect of the P2X7 agonist, BzATP, on intracellular oxidants, transmembrane pores and cell Figure Fluorescence intensities (F 2. Effect of the P2X7 agonist, BzATP, on intracellular oxidants, transmembrane pores and cell DCF) are plotted as the percentage of the control value. (B) Effect of various viability in capillaries located within microvascular complexes freshly isolated from the rat retina. (A) additives on the increase in F DCF induced by a 600‐s exposure to 100 μM BzATP. Additives: A740003 viability in capillaries located within microvascular complexes freshly isolated from the rat retina. Fluorescence intensity of the oxidation‐sensitive dye, dichlorofluorescein (DCF), before and during 7 receptors; UTP (30 μM), a nucleotide whose effect on retinal (200 nM), an inhibitor of P2X (A) Fluorescence intensity of the oxidation-sensitive dye, dichlorofluorescein (DCF), before and during exposure to 100 μM BzATP. Each data point is the mean of 9 regions of interest in retinal capillaries. microvessels includes inhibition of P2X7 pore formation; apocynin (300 μM), an inhibitor of NADPH exposure to 100 µM BzATP. Each data point is the mean of 9 regions of interest in retinal capillaries. Fluorescence intensities (F DCF) are plotted as the percentage of the control value. (B) Effect of various oxidase, and NAC (n‐acetyl cysteine, 100 μM), an antioxidant. Sample size: 47 ± 8. * p = 0.0081; ** p = additives on the increase in F DCFplotted induced by a 600‐s exposure to 100 μM BzATP. Additives: A740003 Fluorescence intensities (FDCF ) are as the percentage of the control value. (B) Effect of various 0.0052; *** p = 0.0009; **** p = 0.0003 (Left panel). Effect of the additives on the BzATP‐induced increase 7 receptors; μM), a nucleotide effect on retinal (200 nM), an inhibitor of P2X additives on the increase in F induced by aUTP 600-s(30 exposure to 100 µMwhose BzATP. Additives: A740003 DCF in the percentage of capillary cells containing the 629 Da dye, YO‐PRO‐1, whose entry is indicative of microvessels includes inhibition of P2X 7 pore formation; apocynin (300 μM), an inhibitor of NADPH (200 nM), an inhibitor of P2X receptors; UTP (30 µM), a nucleotide whose effect on retinal microvessels the presence of transmembrane pores. Sample size: 14 ± 6. * p = 0.0014; ** p